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Expert Views :: Hiffing And Huffing Along The Inca Trail Barbara S. Beckman Environmental challenges were never so abruptly felt by this scientist until a recent trip to the Andes. For years I’d studied the signal transduction pathways critical for hypoxia-regulated erythropoietin production at the biochemical and molecular level (Figueroa et al., 2002). As a pharmacologist I was proud of the "translation" of my favorite hormone, erythropoietin, to the status of a drug with very few side effects. This development was a testament to the pioneers in the field, Eugene Goldwasser of the University of Chicago, and James Fisher of Tulane University (Miyake et al., 1977; Fisher, 2003), among others. Once the hormone was purified by sheer brute force it didn’t take the biotechnology community long to prepare extraordinary amounts of recombinant erythropoietin to treat the anemia of chronic renal failure. In spite of this enormous success the mechanism(s) underlying the tight control of erythropoietin gene expression by oxygen remained enigmatic. What we couldn‘t envision early on was how the availability of the cloned erythropoietin gene, together with the demonstration that particular hepatocellular carcinoma-derived cell lines produced erythropoietin in an oxygen-regulated manner in tissue culture, set the stage for a molecular approach to dissection of the oxygen responsive signal pathway starting with the gene itself. The importance of this pathway is suggested by its presence in nearly all cells within nearly all higher eukaryotes from flies and worms to man. The physiological responses to the stress on the oxygen transport system of creatures used to sea level conditions (below sea level here in New Orleans) include increased heart rate, increased ventilation rate as well as hematological alterations. Failure to adapt can lead to serious medical conditions such as high-altitude pulmonary edema. Despite these obstacles, travelers such as myself, continue to visit the mountains. The Peruvian city of Cusco, at 3350 meters in the Andes and at a PO2 of about two-thirds sea level (Hochachka and Rupert, 2003), is the gateway to the magnificent Inca ruins of Machu Picchu.  Machu Picchu, photographed by Barbara Beckman, 2003 Indigenous populations such as the Quechua of Peru manage to deal with reduced levels of oxygen and have done so for centuries. Scientists have been fascinated with their ability to deal with reduced levels of oxygen (see John West’s book "The High Life" for an entertaining and detailed history of the field). Included in the acute response of lowlanders exposed to hypobaric hypoxia is a robust increase in the synthesis of erythropoietin (Epo), Epo then stimulates the maturation of red blood cells (RBCs). These cells are responsible for the transportation of O2 in the blood and the resultant increase in blood O2-carrying capacity compensates for reduced O2 availability. Erythropoiesis generates new RBCs, both to compensate for routine cell turnover and to allow adaptive increases in hematocrit, such as the response to anemia, inflammation, or hypoxia. In travelers to high altitude, hypobaric hypoxia is detected by O2-sensor-containing cells located in peritubular interstitial cells in the kidney. Once hypoxia is detected, an increased plasma Epo concentration and subsequent elevated hematocrit results compensating for the reduced O2 availability in the inspired air. After some days of acclimatization to hypoxia there is a blunting of the homeostatic response and Epo synthesis drops even though the triggering condition (hypobaric hypoxia) remains unchanged. This blunting of the O2-sensing system is even more pronounced in the Quechua natives who live at high altitudes on the Andean altiplano. It is unclear how this long-term adaptation occurs, but the answer may be found in the O2-sensing mechanism itself. Many of the molecular details have been elucidated over the last decade beginning with the discovery of a transcription factor called hypoxia-inducible factor-1, a dimeric transcription factor composed of a hypoxia regulated subunit (HIF-1a, HIF-2a or HIF-3a) and an oxygen-independent subunit (HIF-1b, also known as aryl hydrocarbon receptor translocator (ARNT) (Wang et al., 1995). HIF-1a subunits are constantly being produced by the cell, but are rapidly degraded under normoxic conditions. Under hypoxic conditions, degradation is blocked and the factor accumulates, initially in the cytoplasm and then in the nucleus where it binds HIF-1b. This process forms a competent transcription factor that can then associate with the highly conserved hypoxia response elements (HREs) within regulatory sequences of hypoxia responsive genes (e.g. Epo) and, in conjunction with other factors, initiate transcription. Degradation of HIF-1a under normoxic conditions is triggered by post-translational events that include proline and asparagine hydroxylation reactions. Co-ordinated function of four different hydroxylases may be a critical component of the O2-sensing system required for hypoxia regulated gene expression. (Hochachka and Rupert, 2003). In addition, long-term adaptation might be related to variations in the gene encoding HIF-1a or in the genes under HIF-1 influence. It is conceivable that sequence modifications in HIF1a or Epo might change binding affinities of these genes and could underlie the fine-tuning of the hypoxia sensitivity of the Epo regulatory system in high-altitude natives. Hochachka and Rupert (2003) did not find variants among the Quechua, suggesting that subtle differences between populations living at high altitude versus those living at lower altitudes must arise by other mechanisms. Actual intracellular O2 concentrations are not known in vivo for most tissues. Since intracellular [O2] is a function of O2 delivery, this means that in vivo it is O2-delivery processes that determine tissue [O2], and thus become the main determinants of the Epo response to hypoxia. In acute hypoxia, mechanisms accommodating the reduced availability of O2 are unable to compensate rapidly enough for the imposed O2 limitation; thus the hypoxic signal displays its maximum effect on the HIF-1:Epo system. In lowlanders exposed to hypobaric hypoxia, acclimation processes affecting O2 delivery can be brought into play to compensate for the external hypoxia and thus minimize the size of the hypoxic signal seen at the cell level. These processes then adequately explain why the Epo response can be attenuated during acclimation. "Finally, in the Quechua, adapted over generations to hypobaric hypoxic altitude conditions, numerous mechanisms including inherently higher blood O2-carrying capacities and also vasodilatory control mechanisms, assure that a given hypobaria leads to a more moderate hypoxia signal to the HIF-1: Epo system at the cell and tissue level, and thus to a blunted HIF-1:Epo response compared to the acute hypoxic response of lowlanders" (Hochachka and Rupert, 2003). Details of the long-term adaptation of the Quechua and other high altitude dwellers will require a comprehensive "systems" biology approach which will no doubt yield an understanding of the fundamental bases for adaptation to a universal stress known as hypoxia.  Photograph by Barbara Beckman, 2003
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